Intercalated cells (IC) respond to pH changes in the blood by increasing or decreasing acid secretion in the kidney collecting duct. Dysfunction of this process results in pathophysiological disorders of different organ systems as the pH of the blood drifts away from its normal value of 7.4. The vacuolar H[+]ATPase (V-ATPase) is central to the acid/base homeostatic function of IC, but how these cells detect environmental cues that allows them to modify proton secretion appropriately remains a mystery. The existence of an acid or bicarbonate sensor in the kidney has long been suggested, but the identity of this detection system and how such a system would transmit signals to modify the acid/base transporting machinery of ICs remain to be determined. Based on work carried out in the previous funding period, we propose here that the soluble adenylate cyclase (sAC) is the much sought after renal acid/base sensor. This protein generates the second messenger cAMP upon direct stimulation by bicarbonate ions. It is, therefore, ideally suited for a bicarbonate/CO{2} sensing role in IC. We hypothesize that cAMP generated by the sAC sensor in response to acid/base cues can modify the acid secretory capacity of intercalated cells. We propose that V-ATPase and sAC are partners in a localized signaling process that modulates targeting and trafficking of the V-ATPase in specific membrane microdomains to regulate intercalated cell function, and renal proton secretion. Our aims are: 1) To characterize the role of sAC in the regulation of V-ATPase mediated proton secretion by renal epithelial cells and 2) To determine whether V- ATPase and cytoskeletal proteins (actin, gelsolin, drebrin, nadrin and myosin VI) form a local micro-complex that regulates V-ATPase membrane accumulation and proton secretion in IC. The studies will use a multidisciplinary approach including unique animal models, isolated fluorescence-sorted intercalated cells, and cell cultures, as well as imaging technologies including static and real-time confocal microscopy to follow V- ATPase trafficking. Assays of vesicle acidification, ATPase activity and proton-selective self-referencing microelectrodes will monitor the functional expression of V-ATPase in endosomes and at the plasma membrane. Fluorescence (Forsman) resonance energy transfer (FRET) and protein-protein interaction assays will dissect whether V-ATPase subunits interact with sAC and/or cytoskeletal proteins during stimulation of proton secretion. In vitro assays, mutational analysis and phosphoproteomics will address the role of cAMP/PKA mediated V-ATPase phosphorylation in these interactions. We propose that the V-ATPase is a central partner in a localized, multi-protein complex that senses and responds to prevailing acid/base conditions by modulating the V-ATPase dependent acidification mechanism in intercalated cells.